phylogenetic relationships and biogeographical patterns in circum

RESEARCH ARTICLE Open Access

Phylogenetic relationships and biogeographicalpatterns in Circum-Mediterranean subfamilyLeuciscinae (Teleostei, Cyprinidae) inferred fromboth mitochondrial and nuclear dataSilvia Perea1*, Madelaine Böhme2, Primož Zupančič3, Jörg Freyhof4, Radek Šanda5, Müfit Özuluğ6, Asghar Abdoli7,Ignacio Doadrio1

Abstract

Background: Leuciscinae is a subfamily belonging to the Cyprinidae fish family that is widely distributed inCircum-Mediterranean region. Many efforts have been carried out to deciphering the evolutionary history of thisgroup. Thus, different biogeographical scenarios have tried to explain the colonization of Europe andMediterranean area by cyprinids, such as the “north dispersal” or the “Lago Mare dispersal” models. Most recently,Pleistocene glaciations influenced the distribution of leuciscins, especially in North and Central Europe. Weighingup these biogeographical scenarios, this paper constitutes not only the first attempt at deciphering themitochondrial and nuclear relationships of Mediterranean leuciscins but also a test of biogeographical hypothesesthat could have determined the current distribution of Circum-Mediterranean leuciscins.

Results: A total of 4439 characters (mitochondrial + nuclear) from 321 individuals of 176 leuciscine speciesrendered a well-supported phylogeny, showing fourteen main lineages. Analyses of independent mitochondrialand nuclear markers supported the same main lineages, but basal relationships were not concordant. Moreover,some incongruence was found among independent mitochondrial and nuclear phylogenies. The monophyly ofsome poorly known genera such as Pseudophoxinus and Petroleuciscus was rejected. Representatives of bothgenera belong to different evolutionary lineages. Timing of cladogenetic events among the main leuciscinelineages was gained using mitochondrial and all genes data set.

Conclusions: Adaptations to a predatory lifestyle or miniaturization have superimposed the morphology of somespecies. These species have been separated into different genera, which are not supported by a phylogeneticframework. Such is the case of the genera Pseudophoxinus and Petroleuciscus, which real taxonomy is not wellknown. The diversification of leuciscine lineages has been determined by intense vicariant events following thepaleoclimatological and hydrogeological history of Mediterranean region. We propose different colonizationmodels of Mediterranean region during the early Oligocene. Later vicariance events promoted Leuciscinaediversification during Oligocene and Miocene periods. Our data corroborate the presence of leuciscins in NorthAfrica before the Messinian salinity crisis. Indeed, Messinian period appears as a stage of gradually Leuciscinaediversification. The rise of humidity at the beginning of the Pliocene promoted the colonization and posteriorisolation of newly established freshwater populations. Finally, Pleistocene glaciations determined the currentEuropean distribution of some leuciscine species.

* Correspondence: [email protected] Nacional de Ciencias Naturales-CSIC. Department of Biodiversity andEvolutionary Biology. José Gutiérrez Abascal 2, 28006 Madrid. SpainFull list of author information is available at the end of the article

Perea et al. BMC Evolutionary Biology 2010, 10:265http://www.biomedcentral.com/1471-2148/10/265

© 2010 Perea et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

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BackgroundMediterranean freshwater fish fauna is characterized bya relatively low number of fish families, with most ofthe species belonging to Cyprinidae [1,2]. In effect, thisfamily is among the most speciose families of freshwaterfishes and likely to be one of the largest vertebratefamilies [3]. The family Cyprinidae also features a rela-tively high number of endemic species on the Mediter-ranean slope [2] and its wide biological and ecologicalplasticity has bestowed this group an important role inbiogeographical models [4-7]. As dispersion mechanismsare highly restricted in primary freshwater fishes [8],their distributions are directly related to paleogeographyand relationships among different areas [9-11]. Thus,phylogenetic relationships among evolutionary lineagesreflect the history of cyprinids within the Mediterraneanregion.The classification of cyprinids has always been a mat-

ter of controversy and from 2 to 12 subfamilies havebeen recognized depending on author and the morpho-logical traits considered [12-15] and even have beenrecently elevated to family level, being assigned Eur-opean and North American leuciscins and phoxinins tothe new called family Leuciscidae [16,17]. Traditionalmorphology, however, sometimes conflicts with themore recent molecular studies [5,18-23] because somemorphological characters are prone to homoplasy [5]and usually there is unclear homology of morphologicaltraits [24,25]. This determines that recognized mono-phyletic groups are clearly misinterpreted. In this paper,we followed the classification scheme of Saitoh et al.[22] based on complete mitochondrial genomes. Theseauthors consider the subfamily Leuciscinae exclusivelyformed by the Eurasian and North African leuciscins(including the North American genus Notemigonus, asthe only known representative in this region) and pro-mote phoxinins [15] to their own subfamily, Phoxininae,as the sister group of the subfamily Leuciscinae. Todate, however, the nuclear phylogenetic relationships ofcyprinids, and particularly leuciscins, have been littleexplored [26,27].The fossil record of Cyprinidae family in Eurasia [28-30]

suggests its origin in East and Southeast Asia, where great-est generic and species diversity is found [1,3,31]. Thisgroup then colonized Europe for the first time during theOligocene period when the Ob Sea disappeared because ofthe uplifting of the Urals, and reached the Iberian Penin-sula (the westernmost part of Europe) in the late Oligo-cene-early Miocene [32]. The colonization of North Africahas been explained by connections with Iberian and Asianfish faunas during Cenozoic period [33]. Within the familyCyprinidae, the subfamily Leuciscinae may be used to testbiogeographical hypotheses for the Mediterranean basin.

In addition, owing to its long-standing distribution rangein the Circum-Mediterranean area it should be possible tounravel the evolutionary history of this group.Traditional hypotheses have proposed the origin of the

subfamily Leuciscinae in the Mediterranean area and itssubsequent diversification through river captures fromcentral Europe as several waves stretched across a longtime period (from the Oligocene until late Pliocene, 35-1.7 mya) [34,35], following the hydrogeographical andgeological history of the European area [36]. This modelhas been designated as the “north dispersal model” [37].Hypotheses opposing this model have argued that thecolonization of Circum-Mediterranean rivers cannot beexplained by a northern route. The alternative modelproposed is based on leuciscine dispersion during thelacustrine stage of the Mediterranean basin [38], whenthis sea became refilled with fresh water from ParatethysSea during the Messinian salinity crisis [39]. This wouldhave allowed Paratethyan freshwater icthyofauna tocolonize the Mediterranean margins. This hypothesis isdescribed as the “Lago Mare” dispersal model [38]. Thelater opening of the Straits of Gibraltar [40] filled theMediterranean area with marine water, with the subse-quent isolation of the new-formed freshwater popula-tions along with intense vicariant events [41,42].However, the Lago Mare and north dispersal hypothesesare not mutually exclusive and together could haveplayed an important role on dispersal of cyprinids acrossEurope [1,11,43]. On the other hand, the Middle Easthas been considered an important interchange area forfreshwater ichthyofauna during the gradual closing ofthe Tethys Sea [6,44,45]. It is in fact held by someauthors that the Middle Eastern freshwater fauna ismade up of species that came from Asia and morerecently from Euromediterranean ancestors [6,45,46].The basis for this latter proposal is the close affinityfound between Middle Eastern and Euromediterraneancyprinids [6,47]. This region has been also recognized asa center of origin for some Leuciscinae species [6,11,48].Most recently, Pleistocene glaciations influenced the

distribution of Leuciscinae representatives, especially inNorth and Central Europe, where some species becamelocally extinct when the region was covered by ice[35,49]. Later recolonization from eastern refuges suchas Circum-Black Sea drainages has been suggested toexplain the wide distribution of some extant cyprinidspecies [11,50,51]. Although the Mediterranean Peninsu-las and Caspian/Caucasus region are known to haveacted as glacial refuges [52,53], the Iberian, Italian andBalkan Peninsulas were isolated from northern and cen-tral Europe because of the Alps uplift during the Pleisto-cene thus preventing most Mediterranean freshwaterspecies moving northwards. This interpretation explains


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the low level of shared freshwater species betweennorth-central and southern Europe.Weighing up all biogeographical scenarios, some mod-

els have attempted to explain the center of origin andthe main dispersion routes for cyprinids [1,6,11,34-36,47,48,54,1], while others have tried to identify barriersto explain the vicariant patterns observed in cyprinidfishes [4,5,14,25,32,33,37,43,55]. Despite these efforts,the phylogenetic relationships of Circum-Mediterraneanleuciscins and their biogeographical patterns in Mediter-ranean area remain unclear.To obtain reliable information on the mitochondrial

phylogenetic structure of this group, the comprehensivestudy examines mitochondrial DNA in numerous spe-cies of the subfamily Leuciscinae. Indeed, sequences ofthe cytochrome b (cyt b) gene have achieved phyloge-netic resolution in some fish groups [56,57], includingcyprinids [4-7,11,18,20,37,43,48,58]. In turn the cyto-chrome oxidase I (COxI) gene has also proved to be auseful tool for the identification of fish species [59,60].Here we complete the mitochondrial phylogeny of thesubfamily Leuciscinae using nuclear information by ana-lyzing the Recombination Activating Gene 1 (RAG-1)and the Ribosomal Protein Gene S7 (S7). Only a fewprevious molecular studies on cyprinids have yielded anuclear phylogeny of Circum-Mediterranean representa-tives of the subfamily Leuciscinae. Some leuciscinegroups have been examined using nuclear allozymes[10,61,62] or DNA sequences [63-65] approaches andsome phylogenetic relationships have been proposed forcyprinids [66]. However, this paper constitutes the firstattempt at deciphering the nuclear relationships of themain Mediterranean Leuciscinae lineages based onsequences data.The aim of this exhaustive study was to investigate

phylogenetic relationships among the major Circum-Mediterranean Leuciscinae lineages by analyzingsequence variation of both mitochondrial (cyt b andCOxI) and nuclear (RAG-1 and S7) genes. Data wereobtaining for 321 individuals representing 176 species ofthe subfamily Leuciscinae and 9 outgroup species. Thedata were used to test for biogeographical events thatcould have determined the distribution of leuciscins inMediterranean area.

ResultsLeuciscinae phylogenetic performanceOut of a total of 4439 characters obtained, 1786bp cor-responded to mitochondrial DNA and 2653bp tonuclear DNA. Table 1 compares the phylogenetic per-formance of the individual genes under the taxon sam-pling in phylogenetic analyses. ML parameters estimatedusing Modeltest v3.7 [67] are provided in Table 2. Thec2 test for base homogeneity indicated that base fre-quency distributions were always homogenous amongtaxa. The nuclear genes showed much higher consis-tency (CI) and retention (RI) indices, while the mito-chondrial genes offered more parsimony informative(PI) sites. Indeed, the proportion of informative sitesshowed by cyt b gene was the highest (Table 1).

Mitochondrial phylogenetic relationshipsAll mitochondrial analyses generated almost identicaland well-supported topologies. Our discussion thereforefocuses on the more resolved Bayesian tree and theresults of the Maximum likelihood (ML) and MaximumParsimony (MP) analyses are summarized. Bayesian ana-lysis [68,69] has been empirically demonstrated to bethe most efficient character-based method for accuratelyreconstructing a phylogeny [70].According to our molecular mitochondrial cyt b data,

the subfamily Leuciscinae comprises fourteen majormonophyletic lineages. All lineages were supported byhigh posterior probability and bootstrap values (Figure 1).Lineage I was formed by the monotypic North Ameri-

can genus Notemigonus (N. crysoleucas). Lineage II com-prised the widespread genus Alburnus, the monotypicgenera Leucaspius and Anaecypris and the North Afri-can species Pseudophoxinus punicus. Although L. deli-neatus appeared as more related with P. punicus, thisrelationship was not highly supported (Figure 2). Line-age III was formed by the genus Scardinius. Lineage IVcomprised the two species of the Greek genus Tropido-phoxinellus and the two North African species Pseudo-phoxinus chaignoni and P. callensis. Lineage V includedthe genera Squalius, Ladigesocypris (L. ghigii and L. iri-deus) and two species of Petroleuciscus (P. borysthenicusand P. smyrnaeus) (Figure 3). The genus Squalius wassubdivided into two well-differentiated clades. The

Table 1 Comparison of the phylogenetic performance for each individual gene and the combined data set

GENE TOTALCHARACTERS

PARSIMONY INFORMATIVECHARACTERS (in %)

Tv/TsRATE

LENGTHparsimony tree

CI RI HI

CYT B 1140 562 (49.29%) 5.45 8995 0.233 0.769 0.882

COxI 646 242 (37.46%) 0.79 2322 0.199 0.797 0.801

NUCLEAR DATA SET (RAG1+S7) 2647 600 (22.66%) 0.87 2531 0.637 0.795 0.363

COMBINED GENES (Cytb+COxI+RAG1+S7)

4339 1258 (28.37%) 1.94 10913 0.371 0.556 0.729


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“S. cephalus species group” clade, which was in turnstructured into two subclades: the first subcladeincluded central European and Mediterranean specieswhile the second subclade consisted of Anatolian andCaucasian species. The second large Squalius clade wasstructured into three subclades: the Iberian subclade,the Adriatic subclade and the Greece subclade formedby the species S. keadicus. Ladigesocypris were nestedinto the Squalius clade and Petroleuciscus were posi-tioned basal to all species of Squalius. Lineage VI wascomprised of the former genus Chondrostoma s.l., whichhas been recently subdivided into six genera [64]:Achondrostoma, Chondrostoma, Iberochondrostoma,Parachondrostoma, Protochondrostoma and Pseudochon-drostoma. The genera Phoxinellus, Telestes and the Ana-tolian representatives of the genus Pseudophoxinus alsoclustered in this lineage (Figure 4). Phoxinellus andChondrostoma s.l., together represented the sister groupof Telestes and the Anatolian Pseudophoxinus. The Ana-tolian Pseudophoxinus species were represented by twomonophyletic and well-differenciated clades: one ofthem including the species P. alii, P. anatolicus, P. anta-lyae, P. battalgilae, P. crassus, P. elizevetae, P. evliyae, P.fahretinne, P. ninae and one possibly undescribed spe-cies. The second one was formed by P. firati, P. kervillei,P. zekayi and P. zeregi. Lineage VII constituted thegenus Rutilus and lineage VIII contained the generaAbramis, Acanthobrama, Acanthalburnus, Ballerus,Blicca, Mirogrex and Vimba, together with the Iranianspecies Petroleuciscus persidis. Lineage IX was exclu-sively represented by the genus Alburnoides. Lineage Xwas composed by the genera Aspius and Leuciscus. Line-age XI was comprised only by the species Pseudophoxi-nus egridiri. Lineage XII was formed by the genusPachychilon. Lineage XIII comprised the genera Pelasgusand Delminichthys. Finally lineage XIV was formed bythe monotypic genus Pelecus, which was the first lineageto split.The cytochrome oxidase I gene (COxI) rendered the

same main lineages as cytochrome b but also detected a

deep politomy among all lineages (Figure 5). Thus,excluded Pelecus basal to the remaining leuciscinelineages with regarding to the phylogenetic relationshipsbetween different lineages, deep relationships betweenthem were not well solved with mitochondrial markers.Moreover, some incongruence was found among cyt b

and COxI topologies, such as Pseudophoxinus punicus,which was more related with P. callensis than with theAlburninae group, as well as Pachychilon, which wascloser to Alburnoides lineage than to Pelasgus-Delmi-nichthys and Pseudophoxinus egridiri lineages. On theother hand, the Lineages II (Alburnus, Anaecypris, Leu-caspius and Pseudophoxinus punicus), III (Scardinius)and IV (Pseudophoxinus callensis, P. chaignoni, Tropido-phoxinellus) were clustered together and highly supportwith cytochrome b, but were unrelated with COxI gene.

Nuclear phylogenetic relationshipsAlthough the number of species is lesser in nuclear ana-lyses than in mitochondrial one, the topologies of bothmitochondrial and combined nuclear genes (RAG1 +S7) support the identical major lineages. However, deeprelationships among lineages were not solved withnuclear markers (Figure 6). Even within some lineagesas number VI (Chondrostoma s.l., Telestes and AnatolianPseudophoxinus) basal relationships were not solved. Asoccurred in COxI topology, the lineages II, III and IVwere unrelated with nuclear genes. Moreover, someincongruence was found between mitochondrial andnuclear data. Such is the case of Pachychilon, which wasnot related with Pelasgus-Delminichtys-Pseudophoxinusegridiri. In turn, P. egridiri was nested, and highly sup-port, within Pelasgus-Delminichthys lineage.

Phylogenetic relationships with all-genes data matrixCombined data analysis recovered the major lineagesand increased their support (posterior probabilities andbootstrap values higher than only mitochondrial andnuclear analysis) (Figure 7). Indeed, this effect can beexplained because large number of variable characters

Table 2 Parameters of ML analyses estimated with Modeltest under Akaike criterion

Cytb COxI RAG1 S7

Model selected by Modeltest(AIC criteria)

GTR+I+G GTR+I+G K81+G K81+G

Rates substitution matrix [A-C] = 0.61 [A-C] = 1.00 [A-C] = 1.93 [A-C] = 1.00

[A-G] = 26.57 [A-G] = 17.05 [A-G] = 5.09 [A-G] = 1.56

[A-T] = 0.35 [A-T] = 1.00 [A-T] = 1.23 [A-T] = 0.64

[C-G] = 1.62 [C-G] = 1.00 [C-G] = 0.54 [C-G] = 0.64

[C-T] = 6.60 [C-T] = 9.43 [C-T] = 5.09 [C-T] = 1.56

[G-T] = 1.00 [G-T] = 1.00 [G-T] = 1.00 [G-T] = 1.00

Assumed proportion of invariable sites (I) 0.36 0.57 0 0

Alpha (G) 0.65 0.98 0.46 1.02


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Figure 1 Phylogenetic tree rendered by Bayesian analysis of the mitochondrial cytochrome b data set. Numbers above branches meansposterior probabilities of BI/Bootstrap values of ML/Bootstrap values of MP.


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can improve the accuracy of reconstructed relationships[71]. Nevertheless, the topology of combined data wasvery similar to that obtained by cytochrome b, due tothe higher number of informative-parsimony charactersof this gene respect to the others (Table 1).

Divergence timeA relaxed molecular clock was performed based onmitochondrial cytochrome b gene (not showed) and all-genes data set. Our analysis supports the divergence of

the main lineages occurred in Late Oligocene-EarlyMiocene. The timing of splitting-events of the Circum-Mediterranean leuciscine lineages is reported in Figure8.

DiscussionPhylogenetic relationships of Leuciscinae lineagesAlthough our study is focused on the Circum-Mediter-ranean Leuciscinae representatives, the inclusion of themajority of west Paleartic lineages allowed a more

Figure 2 Phylogenetic tree rendered by Bayesian analysis of the mitochondrial cytochrome b gene for Alburnus-Anaecypris-Leucaspius-Pseudophoxinus punicus lineage. Numbers above branches means posterior probabilities of BI/Bootstrap values of ML/Bootstrap values of MP.


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Figure 3 Phylogenetic tree rendered by Bayesian analysis of the mitochondrial cytochrome b gene for Squalius-Petroleuciscus lineage.Numbers above branches means posterior probabilities of BI/Bootstrap values of ML/Bootstrap values of MP.


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Figure 4 Phylogenetic tree rendered by Bayesian analysis of the mitochondrial cytochrome b gene for Chondrostoma s.l.-Phoxinellus-Telestes-Anatolian Pseudophoxinus lineage. Numbers above branches means posterior probabilities of BI/Bootstrap values of ML/Bootstrapvalues of MP.


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Figure 5 Phylogenetic tree rendered by Bayesian analysis of the mitochondrial cytochrome oxidase I gene. Numbers above branchesmeans posterior probabilities of BI/Bootstrap values of ML/Bootstrap values of MP.


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Figure 6 Phylogenetic tree rendered by Bayesian analysis of the nuclear dataset (RAG1+S7 genes). Numbers above branches meansposterior probabilities of BI/Bootstrap values of ML/Bootstrap values of MP.


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Figure 7 Phylogenetic tree rendered by Bayesian analysis of the all-genes data set (Cytochrome b-COxI-RAG1+S7 genes). Numbersabove branches means posterior probabilities of BI/Bootstrap values of ML/Bootstrap values of MP.


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accuracy resolution of their phylogenetic framework,and subsequently of their evolutionary history. We havebased our phylogenetic discussion in the all-genes topol-ogy, which was the most solved and supported tree andsummarizes the information of all analyzed genes.Although some studies have tried to explain the phy-

logenetic relationships of Mediterranean leuciscins[4,5,18,37,43,63,64], this study is the first molecularapproximation to the phylogenetic placement of thethree very poorly studied, small size North African Pseu-dophoxinus species: P. callensis, P. chaignoni and P.punicus. We have demonstrated here that none of this

three species belong to the real genus Pseudophoxinus.Morphological approaches have already hypothesized aclose relationship among P. callensis and P. chaignoni inrelation with P. punicus [72]. Our molecular phylogenycorroborate this later affirmation, and whereas P. callen-sis and P. chaignoni were closely related, and in turnclustered with the genus Tropidophoxinellus, P. punicuswas nested within the Alburnus and Leucaspius group.The phylogenetic positions of the North African speciesP. punicus and the genera Leucaspius and Anaecyprisstrongly suggest that these species belong to the lineageof Alburnus. The inclusion of these genera turns into

Figure 8 Timing of the major cladogenetic events in leuciscin lineages based on a relaxed molecular clock and in all-genes data set.Black numbers represent posterior probability values for BI (** symbol means a value of posterior probability equal to 100). Red numbersrepresent divergence times and their HPD 95% confidence intervals.


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paraphyletic the genus Alburnus. For this reason, thisissue opens the question about whether the groupformed by Alburnus baliki, A. orontis and A. sellal fromAnatolia and Levant should represent an independentgenus or if the monotypic miniature Anaecypris andLeucaspius should be included into Alburnus. The inclu-sion of P. punicus into the Alburnus lineage has beenalready proposed based on morphology [72,73], as wellas previous morphological [18,73] and molecular studies[5] have already hypothesized a close relationship ofAnaecypris and Leucaspius with Alburnus. Moreover, asother dwarf leuciscine, Anaecypris also have been placedin Pseudophoxinus or Phoxinellus by some authors[74,75]. As in other small sized and specialized cyprinidsas the Leuciscinae Delminichthys [63] and Pelasgus [2]or the Rasborinae Paedocypris [23], synapomorphiccharacters seem to have been overshadowed by morpho-logical changed connected to miniaturization. However,further specific studies are required to solve these ques-tions. In turn, P. callensis and P. chaignoni are heretransferred provisionally to the genus Tropidophoxinel-lus (See Table 3).The real genus Pseudophoxinus (type species P. zeregi)

comprises the Anatolian and Levant species, which areclustered in the same lineage that Telestes (its sistergroup), Phoxinellus and the six new genera describedfor the former genus Chondrostoma s. l.: Achondros-toma, Chondrostoma, Iberochondrostoma, Parachondros-toma, Protochondrostoma and Pseudochondrostoma [64].Within the Anatolian Pseudophoxinus two well-differen-tiated groups are recognized: one of them is the groupformed by the complex of species inhabiting CentralAnatolia (P. alii, P. anatolicus, P. antalyae, P. battalgi-lae, P. crassus, P. elizavetae, P. evliyae, P. fahettini, P.ninae and one possibly undescribed species) and theother one from the Levant (P. firati, P. kervillei, P. zeregiand P. zekayi). Relationships of Pseudophoxinus werenot in agreement with morphological studies [76,77].We also demonstrate here that P. egridiri forms a sin-

gle group related to the Delminichtys-Pelasgus lineageinstead of the rest of Pseudophoxinus species. Thehypothesis that P. egridiri is not related to Pseudophoxi-nus was already proposed and suspicions that it mightbe more related to the genus Phoxinus have been for-mulated [78]. However, our data rejected this hypothesisand demonstrated that P. egridiri constitutes an inde-pendent phylogenetic lineage. All the species of Delmi-nichthys-Pelasgus lineage are dwarf cyprinids mostlycharacterized by reductive traits but also have someown synapomorphic characters [63,76]. They have beenerroneously included in the genus Pseudophoxinus orPhoxinellus [2,63,79]. Indeed, Pseudophoxinus was con-sidered to be a subgenus of Phoxinellus for many dec-ades [76].

The genus Telestes is closely related to the main groupof Pseudophoxinus and we also support the position ofTelestes croaticus, T. fontinalis and T. metohiensis intothis genus as were previously pointed out [63]. Thesespecies were previously placed in the genus Phoxinellus[79].The genus Squalius is widely distributed throughout

Europe and is highly diversified in the Mediterraneanarea and three groups have been identified within thisgenus [32]: A Mediterranean group composed of smallspecies from southern Spain, Central Italy, SouthernGreece and the Balkans; an Euroasiatic group that iswidely distributed throughout central-east Europe, Asiaand the north of Mediterranean area and the Para-thethys group, which includes species around the Blacksea and Anatolia. Our phylogeny supports these groups,but we have complemented the taxonomical sampling,finding that S. illyricus, S. microlepis, S. svallize and S.tenellus also are included within the Mediterraneangroup reported by Sanjur et al. [32], as well as someAnatolian species in the Euroasiatic group. Furthermore,within the Mediterranean group a geographical structureis observed: an Iberian subgroup, a second Italo-Adriaticsubgroup, and a third Greek subgroup (Peloponnesus),which is not found in the “S. cephalus group”. As sameas Chondrostoma s. l. phylogenetic relationships betweenall of these subgroups were not solved, showing basalpolitomies rather than bifurcating relationships. Con-cerning to “Squalius cephalus“ complex, an underesti-mation on the diversity of this group is observed, afurther taxonomic review is required, especially inEuphrates drainage. In contrast to the remaining Squa-lius species, “S. cephalus“ complex did not show a geo-graphical structure; thus, Greek Squalius species did notform a monophyletic group due to Iberian S. laietanuswas closer to Greek S. orpheus [5,32,37] and Adriatic S.squalus was the sister group of Greek S. prespensis.However, phylogenetic relationships within Greek Squa-lius were in agreement with allozymic [10] and mito-chondrial [80] previous studies.The genus Petroleuciscus (the Squalius Paratethys

group recognized by Sanjur et al. [32]) comprises sixpoorly known species: P. borysthenicus (type species), P.kurui, P. smyrnaeus, P. persidis, P. squaliusculus and P.ulanus [81]. This study represents the first molecularapproach that takes into account three species of Petro-leuciscus. Petroleuciscus turn out to represent a group ofunrelated taxa. Thus, Petroleuciscus borysthenicus and P.smyrnaeus are two closely related species from theAegean and Black sea basins with relationships to thegenera Ladygesocypris and Squalius. Interestingly, Leu-ciscus aphipsi from the northern Caucasus also belongsto this group, being a morphologically very unspecia-lized species with still many plesiomorphic characters


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Table 3 Taxonomy of subfamily Leuciscinae based on the phylogenetic results obtained in this study and lineagesthat need further review

Previous Taxonomy Proposed taxonomy after this study

Genus Notemigonus (Lineage I)

N. crysoleucas [152] N. crysoleucas

Genus Alburnus (Lineage II)

Al. alburnus [2]; Al. arborella [2]; Al. belvica [2]; Al. demiri [153]; Al. escherichii[154]; Al. filippii [154]; Al. hohenackeri [2]; Al. macedonicus [2]; Al. mento [2]; Al.orontis [154]; Al. sarmaticus [2]; Al. thessalicus [2]

Without changes

Alburnus populations from Kizilimark R. Alburnus sp. 1

Alburnus populations from Stryrmon R. Alburnus sp. 2

Al. sellal [154]; Al. baliki [155]; Al. kotschyi [156]; Al. orontis [154] A new genus should be described for these species to avoid theparaphyletic status of Alburnus (Type species: A. alburnus)

Genus Anaecypris (Lineage II)

An. hispanica [2] An. Hispanica

Genus Leucaspius (Lineage II)

L. delineatus [2] L. delineatus

Genus Pseudophoxinus (Lineage II)Ps. punicus [157]

A new genus should be described for this species because realPseudophoxinus (type species P. zeregi) belong to a different lineage

Genus Scardinius (Lineage III)

Sc. acarnanicus [2]; Sc. erythrophthalmus [2]; Sc. graecus [2]; Sc. hesperidicus [2];Sc. plotizza [2]; Sc. scardafa [2]

Without changes

Genus Tropidophoxinellus (Lineage IV) Genus Tropidophoxinellus

Tr. hellenicus [2]; Tr. spartiaticus [2] Without Changes

Genus Pseudophoxinellus

Ps. callensis [77] Tr. callensis

Ps. chaignoni [77] Tr. chaignoni

Genus Petroleuciscus (Lineage V) Without changes

Pe. borysthenicus [2], Pe. smyrnaeus [2] Genus Petroleuciscus

Genus Squalius

Sq. aphipsi [2] Pe. aphipsiNecessary to analyze all species considered as belonging toPetroleuciscus (P. kurui, P. squaliusculus, P. ulanus)

Genus Ladigesocypris (Lineage V) Genus Squalius

La. ghigii [2]La. irideus [2]

Sq. ghigiiSq. irideus

Genus Squalius (Lineage V)

Sq. albus [2]; Sq. aradensis [2]; Sq. carolitertii [2]; Sq. castellanus [158]; Sq.cephalus [2]; Sq. illyricus [2]; Sq. keadicus [2]; Sq. kosswigi [159]; Sq. kottelati[159]; Sq. laietanus [2]; Sq. lepidus [2]; Sq. lucumonis [2]; Sq. malacitanus [2]; Sq.microlepis [2]; Sq. moreoticus [2]; Sq. orientalis [2]; Sq. orpheus [2]; Sq.pamvoticus [2]; Sq. peloponensis [2]; Sq. prespensis [2]; Sq. pyrenaicus [2]; Sq.squalus [2]; Sq. svallize [2]; Sq. tenellus [2]; Sq. torgalensis [2]; Sq. valentinus [2];Sq. vardarense [2]; Sq. zrmanjae [2]

Without changes

Squalius populations from Southern Spain Squalius sp

Squalius populations from Aksheir L. Turkey Squalius sp_Euphrates

Squalius populations from Euphrates drainage Squalius sp_Aksheir

Squalius populations from Rama L. BiH Squalius aff. vardarensis

Squalius populations from Manikiotico R. Squalius cf. orpheus

Genus Achondrostoma (Lineage VI) Achondrostoma

Ach. arcasii [2]; Ach. occidentale [2]; Ach. salmantinum [158]; Ach. oligolepis [2]Achondrostoma populations from NE Spain

Without changesAchondrostoma sp

Genus Chondrostoma (Lineage VI)

Ch. angorense [160]; Ch. cyri [160]; Ch. holmwoodii [161]; Ch. knerii [2]; Ch.meandrense [160]; Ch. nasus [2]; Ch. oxyrhynchum [2]; Ch. phoxinus [2]; Ch.prespensis [2]; Ch. regium [160]; Ch. soetta [2]; Ch. vardarense [2]Ch. olisiponensis [162]

Without changesIberochondrostoma olisiponensis


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Table 3 Taxonomy of subfamily Leuciscinae based on the phylogenetic results obtained in this study and lineagesthat need further review (Continued)

Genus Iberochondrostoma (Lineage VI)

Ib. almacai [2];; Ib. lemmingii [2]; Ib. lusitanicum [2]; Ib. oretanum [2] Without changes

Genus Parachondrostoma (Lineage VI)

Pa. arrigonis [2]; Pa. miegii [2]; Pa. toxostoma [2]; Pa. turiense [2] Without changes

Genus Phoxinellus (Lineage VI)

Ph. alepidotus [2]; Ph. dalmaticus [2]; Ph. pseudalepidotus [2] Without changes

Genus Protochondrostoma (Lineage VI)

Pr. genei [2] Without changes

Genus Pseudochondrostoma (Lineage VI)

Pse. duriense [2]; Pse. polylepis [2]; Pse. willkommii [2] Without changes

Genus Pseudophoxinus (Lineage VI)

Ps. alii [77]; Ps. anatolicus [154]; Ps. antalyae [154]; Ps. battalgilae [154]; Ps.crassus [154]; Ps. elizavetae [77]; Ps. evliyae [163]; Ps. fahrettini [163]; Ps. firati[77]; Ps. kervillei [164]; Ps. ninae [63]; Ps. zekayi [77]; Ps. zeregi [165]

Without changes

Pseudophoxinus populations from Eflatun Pinari spring. Turkey Pseudophoxinus sp_EflatunPinari

Genus Telestes (Lineage VI)

Te. alfiensis [63]; Te. beoticus [2]; Te. croaticus [2]; Te. fontinalis [2]; Te.metohiensis [2]; Te. montenegrinus [2]; Te. muticellus [2]; Te. pleurobipunctatus[2];Te. polylepis [2]; Te. souffia [2]; Te. turskyi [2]; Te. ukliva [2]Telestes populations from Dreznica. Croatia

Without changesTelestes sp.

Genus Rutilus (Lineage VII)

Ru. aula [2]; Ru. basak [2]; Ru. frisii [2]; Ru. heckeli [2]; Ru. ohridanus [2]; Ru.panosi [2]; Ru. pigus [2]; Ru. prespensis [2]; Ru. rubilio [2]; Ru. rutilus [2]; Ru.ylikiensis [2]

Without changes

Genus Abramis (Lineage VIII)

Ab. brama [2] Without changes

Genus Acanthobrama (Lineage VIII) Genus Acanthobrama

Ac. lissneri [166]; Ac. marmid [154] Without changes

Genus Acanthalburnus (Lineage VIII)

Aca. microlepis [154] Ac. microlepis

Genus Petroleuciscus (Lineage VIII)

Pe. persidis [167] Ac. Persidis

Genus Ballerus (Lineage VIII)

Ba. ballerus [2]; Ba. sapa [2] Without changes

Genus Blicca (Lineage VIII)

Bl. bjoerkna [2] Without changes

Genus Mirogrex (Lineage VIII)

Mi. terrasanctae [164] Without changes

Genus Vimba (Lineage VIII) Genus Vimba

Vi. melanops [2]; Vi. vimba [2] Without changes

Genus Acanthobrama

Ac. mirabilis [154] (Vi. Vimba by synonymy [6]) V. mirabilis

Genus Alburnoides (Lineage IX)

Alb. bipunctatus [2]; Alb. prespensis [2]; Alb. bipunctatus [2] Without changes

Genus Leuciscus (Lineage X) Genus Leuciscus

Le. idus [2]; Le. latus [167]; Le. leuciscus [2]; Le. schdmidti [46]; Le. walecki [168] Without changes

Genus Aspius (Lineage X)

As. aspius [2]; Le. aspius

As. vorax [2] Le. vorax

Genus Pseudophoxinus (Lineage XI)P. egridiri [154]

A new genus should be described for this species because realPseudophoxinus (type species P. zeregi) belong to a different lineage


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[82,83]. Although this species was transferred from thegenus Leuciscus into the genus Squalius [2,84], our out-come demonstrates that it is more related to the genusPetroleuciscus than to Squalius and it is therefore trans-ferred to Petroleuciscus here (See table 3). Iranian Petro-leuciscus persidis was initially described as a species ofPseudophoxinus [85], later transferred to the genus Leu-ciscus [44] suspected to be related to P. smyrnaeus (for-mer considered as L. smyrnaeus) [86] and then includedin Petroleuciscus [81]. These relationships are inconsis-tent with our molecular data, which strongly supportclose affinities of P. persidis with the genus Acantho-brama, also pointing on the difficulties of morphologicalcharacterization of miniaturized cyprinids [23,63].Therefore, this species is transferred to Acanthobramaas A. persidis (See table 3). Furthermore, our data sup-port the synonymization of the genus Acanthalburnuswith Acanthobrama, both genera just distinguished bythe two vs. one rows of pharyngeal teeth [46]. Ourresults demonstrate also that both species of the highlyspecialized, predatory genus Aspius belong to Leuciscus.Therefore, these species are transferred to Leuciscus asL. aspius and L. vorax (See table 3).The position of the genus Ladygesocypris (L. ghigii and

L. irideus) nested in or close to the Squalius clade wasalready reported for mitochondrial data [6,32] and hereis also supported by nuclear markers. Ladygesocypriswas recently included into the genus Pseudophoxinus[77], a hypothesis that has not molecular support. How-ever, its transfer to Squalius is supported by our data(See table 3).With regards to Scardinius, this genus have been con-

sidered the sister group of Tropidophoxinellus [5,87] buta close phylogenetic relationship among Scardinius andAlburnus has also been hypothesized [5,18]. Our phylo-geny strongly supported the sister relationship amongScardinius and Tropidophoxinellus more than amongScardinius and Alburnus, independently of the NorthAfrican species Pseudophoxinus chaignoni and P. callen-sis belong to the genus Tropidophoxinellus or not. Ournuclear phylogeny showed a low support for a

relationship among Scardinius and Rutilus, as suspectedalready by morphological characters [14]. Whereas mito-chondrial cyt b analysis grouped together the lineages ofScardinius, Tropidophoxinellus-Pseudophoxinus callen-sis-P.chaignoni (transferred here to Tropidophoxinelluscallensis and T. chaignoni) and Alburnus-Anaecypris-Leucaspius-Pseudophoxinus punicus, basal relationshipsamong these lineages were not solved with COxI andnuclear markers, probably due to the slower evolution-ary rate of these genes respect to cyt b one. However,combined data matrix (all genes) analysis recovered thisrelationship. Within the genus Scardinius, our data sup-port some recognized species [2]. S. erythrophthalmus isa Central and Eastern European species, S. scardafaendemic to Lake Scanno in Central Italy [62,88] is clo-sely related to S. hesperidicus, as has been previouslyestablished [62,89]. Scardinius plotizza from the Dalma-tian River Neretva constituted an independent andhighly supported clade.

BiogeographyDivergence time estimates and general biogeographicalpatternsTo decipher evolutionary and biogeographical patternsin the Mediterranean leuciscins, the different authorshave calibrated a molecular clock for the cytochrome bgene using fossil or geological data [5,7,20,37,43,90].Based on fossil calibration, we obtained an evolutionaryrate of 0.4% divergence per lineage per million years(0.8% per pairwise comparison) for cytochrome b, whichdiffers from previous estimates [5,90], and is slightlyslower than formerly proposed for North Americancyprinids based on fossil data (0.5% per lineage per mil-lion year) [91]. The present study is the first to usenuclear markers to estimate the main historical pro-cesses that gave rise to the current phylogeny and distri-bution of the Mediterranean leuciscine lineages. Theaverage evolutionary rate obtained here was 0.2% perlineage per million years for the combined dataset (cyto-chrome b, COxI, RAG1 and S7) (0.4% per pairwise com-parison). Notwithstanding, estimated divergence times

Table 3 Taxonomy of subfamily Leuciscinae based on the phylogenetic results obtained in this study and lineagesthat need further review (Continued)

Genus Pachychilon (Lineage XII)

Pac. macedonicum [2]; Pac. pictum [2] Without changes

Genus Delminichthys (Lineage XII)

D. adpersus [2]; D. ghetaldii [2]; D. jadovensis [2]; D. krbavensis [2] Without changes

Genus Pelasgus (Lineage XIII)

P. laconicus [2]; P. marathonicus [2]; P. minutus [2]; P. prespensis [2]; P.stymphalicus [2]; P. thesproticus [2]

Without changes

Genus Pelecus (Lineage XIV)

P. cultratus [2] Without changes


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were congruent to those predicted by the cytochrome bgene (not shown) and fell within the same confidenceintervals. However, credibility intervals of the combineddataset (i.e. more informative sites in the analysis) werenarrower, suggesting that the combination of severalmarkers may improve the results [92].The complex paleogeography of the Mediterranean

region, with migrating island arcs, fragmenting tectonicbelts and other plate tectonic events have contributed tothe formation of a reticulate biogeographical pattern inwhich the current leuciscine groups formed as geogra-phical barriers appeared and disappeared through time.This reticulate pattern is, however, not reflected by acongruent biogeographical pattern in leuciscine relation-ships based on sister groups within each evolutionarylineage.Origin of leuciscins in Mediterranean inland waters: birth ofthe ParatethysAccording to our molecular evolutionary rate based on arelaxed molecular clock and all-gene database, the mainMediterranean leuciscine lineages arose and diversified inthe Oligocene period (Figure 8). In the context of thewidely accepted origin of cyprinids in Asia, our data donot contradict the north-dispersal model of the firstcyprinid colonization of northern Europe across Siberia[34,35], when the Turgai Straits were closed around theEocene-Oligocene boundary (approximately 34 mya; Fig-ure 9A) and strongly support a gradual colonization ofMediterranean region since Oligocene, as have beenrecently stated [93]. As the fossil record shows, this firstcyprinid colonization event involved phoxinin and gobio-nin lineages [94,95]. However, according to our data, thepresence of old leuciscins in the Balkan/Anatolia area ofpossible Southwest Asian origin could be explained bythe emergence of a huge Balkanian-Anatolian-Iranianlandmass in the Early Oligocene (33 mya) [96]. Continen-tal collisions and tectonic movements along the Alpine-Himalayan orogenic belt, which led to the separation ofthe Paratethys Epicontinental Sea from the TethysOcean, drove this event. We therefore propose an initialleuciscine colonization of Europe from southwesternAsia via the Balkanian-Anatolian-Iranian landmass at thebeginning of the Early Oligocene (Figure 9B), which pre-cisely matches the initial splitting of the most basal leu-ciscine lineages (XI, XII and XIII) from the remainingleuciscins in 32.9 mya (CI 95% 29.6-33.5).Unequivocal fossil proof of this biogeographic hypoth-

esis is hampered by the fact that Oligocene freshwaterdeposits from the Balkanian-Anatolian-Iranian landmassare scarce and not well studied. The oldest fossils thatcould be ascribed to the Leuciscinae have been recov-ered in central Anatolia (Table 4) and have been datedaround the Oligocene-Miocene boundary [97,98]. Thismaterial consists of isolated pharyngeal teeth and

otoliths for which a leuciscine relationship is possible,but not certain.Vicariant events promoting leuciscine diversification in theOligocene: Paratethys isolation and reconnectionDuring most of the Early Oligocene (Solenovian regionalstage), the Tethys and Paratethys Seas remained isolatedby the Balkanian-Anatolian-Iranian landmass. Marineconnections fragmented this landmass around 28 myainto the huge Balkanian and Anatolian islands [96]. Thisvicariant event was probably responsible for splitting ofthe ancient Balkan-Anatolian lineages around the Early-Late Oligocene transition (29.1 mya, CI 95% 27.6-31.4).This main Late Oligocene paleogeographic (Figure 9C)setting persisted until the early Burdigalian 20 to 19mya.By the end of the Oligocene (23 mya) half of all leu-

ciscin lineages (7 out of 14) (Figure 8) had becomeestablished, indicating their rapid diversification duringthe Late Oligocene. During this time, leuciscine fossilsare unknown from the European mainland and wehypothesize that this diversification occurred on theBalkanian-Anatolian-Iranian archipelago. Geodynamicdata substantiating vicariant events in the Late Oligo-cene are rare from this area. For the time being, it maybe speculated that karstic landscapes, similar to thosefound in this region today, could have facilitated thegeographical isolation and speciation process.Vicariant events promoting leuciscine diversification in theEarly-Middle Miocene: the Alpine Orogeny, Dinarid Lakesystems and Gomphotherium landbridgeMore recent paleogeographical events support the split-ting of some leuciscine groups in the Early-Middle Mio-cene, from 20 to 12 mya, giving rise to most of thecurrent genera. This period coincides with a major per-iod in the Alpine orogeny, including the closing of theSlovenian corridor, the extrusion and uplift of the East-ern Alps and substantial microplate rotations and basininversion in the Pannonian-Carpathian-Dinaric realm[99-104].The Slovenian corridor, a marine connection between

the Tethys and Paratethys Seas separated the Balkanian-Anatolian landmass from the rest of Europe during theOligocene and earliest Miocene. This gateway closedaround 20 mya [105] and probably enabled the firstcolonization of Central Europe by leuciscins from theBalkanian-Anatolian landmass (Figure 9D). This view issupported by fossils like the oldest European leuciscinsin sediments from about 19 mya in Germany related toPseudophoxinus and Delminichthys [94,106,107] andfrom 19-18 mya old fossils of Aspius and “Alburnus“[108,109] in the Czech Republic. These paleontologicaldata fit the start of diversification of leuciscins such asAspius (20.5 mya, CI 95% 17.6-21.3) or Alburnuslineages (19.7 mya, CI 95% 16.7-21.9).


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The Alpine orogeny similarly affected the leuciscineichthyofauna of the Mediterranean Peninsulas. Thus, inthe Iberian Peninsula, the Pyrenees promoted the isola-tion of Iberian freshwater fishes during the Mioceneperiod when they took on their current form [110-112].Indeed, all leuciscins from this region are endemicexcept Squalius laietanus, which also inhabits southernFrance. Thus, the Iberian Squalius must have split fromthe Balkan and Italian Squalius 14.6 mya (CI 95% 10.9-14.9). In turn, the endemic genera Achondrostoma,

Iberochondrostoma, Parachondrostoma and Pseudochon-drostoma split from the genus Chondrostoma 9.4 mya(CI 95% 7.9-11.4). The Pyrenees may have also consti-tuted a vicariant barrier between the European and Iber-ian Alburnine (ancestors of Anaecypris, Alburnus and/orLeucaspius), splitting 12.1 mya (CI 95% 9-14.8). Thistiming of the origin of Iberian Leuciscinae lineages iscongruent with the results of previous studies [43,64].In the Apennine Peninsula, the Alpine orogeny may

have also played an important role in isolating its

Figure 9 Map of the main paleogeographic events in Mediterranean region during Late Eocene-Early Miocene periods. Black arrowspoint out marine movements. SC: Slovenian corridor. BL: Balkanian landmass.


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ichthyofauna. Most of Italy was below sea level duringmost of the Miocene [113] and it is consequentlybelieved that the Italian ichthyofauna is of a more recentorigin than that of the remaining Mediterranean Penin-sulas. Although dispersion during the lacustrine phase ofthe Mediterranean Sea has been suggested as the originof the Italian ichthyofauna [38], our data point to anolder origin, probably related to the lateral tectonicextrusion of the eastern Alps between 17 and 11 myathat isolated Italian taxa from north and central Europe.The role of the eastern Alps as a barrier to Italian andAdriatic freshwater ichthyofauna has been previouslyestablished [114,115]. This is the case for Protochondros-toma 11.5 mya (CI 95% 9.5-13.5), or some Rutilus, whichaccording to our mitochondrial data (figure not shown),split 9.7 mya (CI 95% 6.2-10.6) into R. rubilio and 13.2mya (CI 95% 9.4-15.1) into R. pigus. This is the only casein which mitochondrial information has been used to dis-scus date estimation. This can be explained due to weonly incorporated a few representatives of each genera toperform all-gene molecular clock analysis and none ofthese species have been include on all-gene dataset. How-ever, the knowledge of this splitting date for species suchas Protochondrostoma genei or Rutilus rubilio and R.pigus is crucial to show the effect of Alps as a barrier forItalian and Adriatic freshwater icthyofauna.In the Balkan Peninsula, and especially in the Dalma-

tian area, huge lake systems existed between 17 and ~15

mya during the late early Miocene and early MiddleMiocene (e.g. Dinaric Lake system, Lake Skopje; [116]).The fragmentation or disappearance of these lakes couldhave enhanced isolation and promoted speciation. Suchis the case of Delminichthys, which split from Pelasgusin approximately 14 mya (CI 95% 11.0- 20.7). This dat-ing is close to previously estimates (13 mya; [63]) andalso coincides with the splitting of Chondrostoma s.l.from its sister group Phoxinellus isolated in Dalmatia14.6 mya (CI 95% 12.1-16.7). Subsequent to this periodof lakes, the region experienced the onset of compres-sional tectonic stress in the late Serravallian around 12.5mya, initiating a period of uplift of the Dinaric Alps[117]. This orogenic event could have generated barriersfor the Dalmatian Chondrostoma, Squalius and Telestes,which split approximately 9.7 mya (CI 95% 6.2-11.9), 9.2mya (CI 95% 5-10.1) and 12.4 mya (CI 95% 11-16.3)respectively.Another important vicariant event between the Greek

and North African Tropidophoxinellus took place in theEarly Miocene in 17.4 mya (CI 95% 7.9-16.6), indicatingcolonization of Africa by leuciscins well before the Mes-sinian salinity crisis. This finding rejects the laterhypothesis of “Lago Mare dispersal” [38] for the coloni-zation of North Africa by leuciscins, which wouldexplain the colonization of this region due to dispersionduring the lacustrine phase of the Mediterranean Sea(Messinian salinity crisis). The most plausible

Table 4 First fossil occurrence of Leuciscinae in Europe

FOSSIL TAXON RECENT RELATIVES LOCALITY AGE(Ma)

REFERENCE

Leuciscinae indet. Leuciscinae Kilcak (Eskikilcak), central Anatolia 23.5-24.5

[150]

Palaeoleuciscusdietrichsbergensis

Pseudophoxinus, Delminichthys Dietrichsberg, Rhön mountains,Germany

19 [94,106,107]

Aspius laubei Aspius aspius North Bohemian Browncoal Basin,Czech Republic

18-19 [108,109]

Alburnus steindachneri Alburnus chalcoides species group North Bohemian Browncoal Basin,Czech Republic

18-19 [108,109]

Leuciscus sp. Leuciscus s. str. Sofca, Turkey 11-13 Böhme unpubl.,[150]

Scardinius haueri Scardinius ssp. Vösendorf, Austria 10.3 [151]

Leuciscus aff. cephalus Squalius cephalus Lava 2, Greece 6.56 Böhme unpubl.,[150]

“Chondrostoma” sp. Achondrostoma, Pseudochondrostoma, Iberochondrostoma,Parachondtrostoma

Tolosa, Spain 5.33-5.8

Böhme unpubl.,[150]

Scardinius cf.erythrophtalmus

Scardinius erythrophthalmus Ptolemais 1A, Greece 5.31 Böhme unpubl.,[150]

Aspius sp. Aspius aspius Ptolemais 1B, Greece 5.29 Böhme unpubl.,[150]

Chondrostoma sp. Chondrostoma ssp. Ptolemais 2, Greece 5.21 Böhme unpubl.,[150]

Rutilus sp. Rutilus ssp. Tchelopetchene, Sofia Basin, Bulgaria 4-5.3 Böhme unpubl.,[150]


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explanation for vicariance between the Peloponessus andMagreb area is the migration of common ancestors ofTropidophoxinellus from the Balkanian-Anatolian land-mass into North Africa via the Gomphotherium land-bridge established in West Asia around 19 mya (Figure9D). This landbridge separated the Tethyan Ocean intothe Mediterranean Sea and Indian Ocean and allowedthe first continental exchange between Eurasian andAfrican mammals and reptiles [118,119] and the firstcolonization of Africa by barbin cyprinids ([120]; Böhmeunpublished data). Despite fossil leuciscins being as yetunknown from the African Miocene, the data presentedhere suggest that leuciscins probably took part in thisEarly Miocene dispersal event.Of special biogeographic interest is the sister-group

relationship between Notemigonus crysoleucas, the onlyNorth American leuciscine species, and the old Balka-nian-Anatolian Pseudophoxinus egridiri/Pelasgus/Delmi-nichthys lineages, which separated according to our dataaround 20.3 mya (Figure 8). However this relationshiphas to be considered with caution due to its relativelylow support (posterior probability equal to 86). As sta-ted above, a possible sister taxon of the Pseudophoxinusegridiri/Pelasgus/Delminichthys lineages, the fossil genusPalaeoleuciscus, made its oldest fossil appearance in 19mya old sediments of central Europe, indicating a trans-atlantic biogeographic connection. Interestingly, an EarlyMiocene transatlantic colonization of North America bycyprinids was already speculated by Böhme [94] in thecontext of the paleobiogeography of phoxinins. Thisauthor demonstrates a sister-group relationship betweenthe central European Oligocene phoxinin Palaeorutilusand the eastern North American Creek Chub clade ofphoxinins. The data presented here further support suchan idea, and may indicate that eastern North Americawas colonized during Early Miocene times via a transat-lantic route by the ancestors of the leuciscin Notemigo-nus (a species related to Palaeoleuciscus) and the CreekChubs (species related to Palaeorutilus). The paleogeo-graphic details of this hypothesis, however, remainobscure.Vicariant events promoting leuciscine diversification in theUpper MioceneIn this period, the opening of the Aegean Sea was animportant event for leuciscins (Figure 9E), which isthought to have started in the Late Serravallian about 12mya [101] and finished during the Tortonian between10 and 9 mya [121-123]. As a consequence, somegroups of Greek and Anatolian leuciscins diversified,such as Squalius ghigii/irideus from Rhodos Island andSouthwestern Anatolia and Squalius keadicus fromsouthern Greece. These taxa split 8.7 mya (CI 95% 7-13.2). In fact, the paleogeographical history of the

Balkan Peninsula is strongly linked to the paleogeogra-phy of Anatolia since both areas were joined in thesame landmass and isolated from the rest of Europeduring long periods of the Oligocene and Miocene.The North African Pseudophoxinus punicus splits near

the Middle-Late Miocene boundary; also well before theMessinian salinity crisis. Two possible vicariant barrierscould explain its distribution: the opening of the Gibral-tar Straits (splitting of Iberian Anaecypris), which hasbeen recognized as a geographical barrier for somegroups [124], or the opening of the Channel of Sicily(splitting of European Alburnus or Leucaspius). How-ever, both the Gibraltar Straits and the Sicily Channelformed at the Miocene/Pliocene boundary at the end ofthe Messinian salinity crisis [40,125] and our estimateddivergence time for P. punicus is earlier than this date(10.9 mya, CI 95% 6.2-12.2). The vicariant event leadingto the separation of Pseudophoxinus punicus thereforeremains unclear.The MessinianThe Messinian has been postulated as the time of diversi-fication of Mediterranean cyprinids. Hence, the LagoMare stage of the Mediterranean would have enabled themassive dispersion of cyprinids across the basin. Thelater return to marine conditions could have meant a fastspeciation process for cyprinids. This may be reflected inthe deep polytomies found in the phylogeny. Thus, someauthors have defended the Lago Mare hypothesis [38]based on non-resolved polytomies among the mainclades [7,42,54]. However, our phylogeny does not showa critical point of speciation around 5 mya. These pro-cesses took place gradually over time, but preclude anymassive dispersal and speciation during the Messinianperiod, as has been proven for Squalius and Chondros-toma s.l. [37,43,64]. Fossils of the Spanish genera (Achon-drostoma, Iberochondrostoma, Parachondrostoma,Pseudochondrostoma) are recorded first in the late Messi-nian (Table 4), which may indicate dispersal of theirancestors during the Messinian Lago Mare stage.Pliocene and PleistoceneThe beginning of the Pliocene is characterized by a risein humidity in the Mediterranean region [126], whichmay have promoted the diversification of Leuciscinaegenera in different Mediterranean Peninsulas, throughthe possible colonization of newly established freshwaterenvironments after the Messinian salinity crisis. In theIberian Peninsula, tectonic disconnections of present-day Iberian basins took place during the Plio-Pleistocene[127] promoting widespread endorrheism and thusenhancing the process of allopatric speciation, which isreflected in the high species-levels of most Iberian gen-era except the monotypic genus Anaecypris. In turn, thedivergence of some Italian taxa from their Balkanian


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sister-taxa occurred in this period, such as the splittingof Rutilus aula (4.75 mya, CI 95% 1.91-5.02), and Squa-lius lucumonis (3.53 mya, CI 95% 2.13-5.80).In the Adriatic-Balkanian region, even more recent

events explain the great affinities found between theichthyofauna of northern Italy and northern rivers ofthe Balkan Peninsula. These similarities reflect recentcontact between North Italian and North Adriatic-Balk-anian ichthyofauna due to expansion of the Po Riverduring the Last Glacial Maximum as a result of a dropin sea level [128]. This event may have prompted theexchange of many freshwater taxa among Italian andBalkan river systems, and also explains the current dis-tribution of species such as Squalius squalus and simila-rities found between Chondrostoma soetta and theDalmatian species Chondrostoma knerii and C. phoxinus.Similar affinities have been described for freshwater fishspecies from northern Italy and Balkan rivers includingCobitis [129] or Cottus [130] species.On the other hand, Pleistocene glaciations also played

an important role in the current European distributionof the Leuciscinae, and determined a more recent originof some leuciscine taxa after colonization from glacialrefuges such as the Danube basin. However, other riversof the Black Sea basin could have also acted as a glacialrefuge for freshwater ichthyofauna [51]. Some north-central European representatives of leuciscins share awidespread distribution range: Alburnoides bipunctatus,Alburnus alburnus, Chondrostoma nasus, Rutilus rutilus,Scardinius erythrophthalmus and Squalius cephalus.This pattern can be explained by the homogeneity con-ferred by glacial refuges [35,131-133]. Thus, secondaryrecolonization of Western Europe from a Danubianrefuge has been postulated for S. cephalus [131] and C.nasus [34,134]. River captures, river confluences and sealevel lowering have been incriminated in the dispersionof these species [131]. Thus, glacial periods promotedthe expansion and genetic homogenization of these spe-cies across Europe [48,131,135]. Since most central Eur-opean basins were covered by ice during thePleistocene, colonization is the most likely mechanismto explain the expansion of these species. However, theycould not have reached the Mediterranean Peninsulasbecause of the transverse alignment of Alpine Moun-tains, which were well formed in the Pleistocene exceptin the eastern part of the Balkan Peninsula. This areashows the influence of the Danube basin due to theoblique direction of the Dinaric Alps.

ConclusionsMitochondrial and nuclear results demonstrated theexistence of fourteen major phylogenetic leuciscinelineages. However, some incongruence was foundbetween mitochondrial and nuclear markers, possibly

due to the lack of resolution of deep nodes in nuclearphylogeny as well as their slower evolutionary rate.Combined analysis (mitochondrial + nuclear) recoveredthe major lineages and increased their support.With regards to phylogenetic relationships, this study

is the first molecular approximation to the phylogeneticplacement of North African leuciscine species (P. callen-sis, P. chaignoni, and P. punicus). None of these threespecies belong to the real genus Pseudophoxinus, as wellas P. egridiri, and constitute independent lineages. Thereal genus Pseudophoxinus includes species from Anato-lia and Levant. Our phylogeny also demonstrates thatthe genus Petroleuciscus is polyphyletic and as a resultits species involve different leuciscine lineages. It alsopoint out the closer relationship of Squalius aphipsi toPetroleuciscus. As our phylogeny show, the taxonomy ofthe genera Pseudophoxinus and Petroleuciscus is a mat-ter of controversy because of synapomorphic charactersare prone to be overshadowed by morphological changesassociated to miniaturization. New insights in the phylo-genetic relationships of some Squalius species areshowed too, such as some Dalmatian species, as well asthe corroboration by nuclear markers of the phyloge-netic position of Ladigesocypris ghigii/irideus as closerrelated to the genus Squalius.In relation to biogeographical history of Leuciscinae,

the present study is the first to use nuclear markers toestimate main historical processes that gave rise to thecurrent phylogeny and distribution of Circum-Mediter-ranean Leuciscinae. A relaxed molecular clock corrobo-rated the arisen and divergence of the main Leuciscinaelineages during Late Oligocene-Early Miocene. Our datado not contradict the north-dispersal model of the firstcyprinid colonization of Northern Europe across Siberia.However, most of the divergence events were older thanLago Mare dispersal model. We proposed an initialcolonization of Europe from Southwestern Asia via theBalkanian/Anatolian/Iranian landmass at the beginningof the Early Oligocene, which precisely matches theinitial splitting of the most basal leuciscins. Later vicar-iant events as the Paratethys isolation and later recon-nection with Tethys during the Oligocene and theAlpine Orogeny, Dinarid lake systems and Gomphoter-ium landbridges during Miocene promoted Leuciscinaediversification. Our data also corroborate the coloniza-tion of North Africa before the Messinian salinity crisis.In Upper Miocene the opening of Aegean Sea was animportant vicariant event for Anatolian and Greek leu-ciscins. Messinian appears as a stage of gradually Leucis-cinae diversification more than a critical point ofspeciation. The rise of humidity at the beginning of thePliocene may have promoted the diversification of Leu-ciscinae genera due to the colonization of newly estab-lished freshwater environments. Finally, Pleistocene


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glaciations also played an important role in the currentEuropean distribution of some leuciscins.

MethodsSampling, DNA extraction, PCR and sequencingThe complete mitochondrial cytochrome b (total of1140 bp) from 321 specimens belonging to 176 speciesof leusciscins was obtained from Europe, WesternAsia, North Africa and North America (Figure 10). Oftotal individuals, 186 were new sequences for cyto-chrome b and the remaining sequences were acquiredfrom Genbank. Similarly, 146 individuals weresequenced to obtain a fragment of the mitochondrialCytochrome oxidase I gene (646 bp). For the nuclearphylogenetic analysis, a subset of 101 individuals wasselected from mtDNA lineages and sequenced for boththe nuclear genes RAG1 (1473 bp) and the first intronof the ribosomal protein S7 (1112 bp total alignmentincluding gaps). DNA voucher specimens were depos-ited at the collection of the Museo Nacional de Cien-cias Naturales (Madrid, Spain). List of individuals,localities and GenBank Accession numbers arereported in Additional file 1.For phylogenetic analyses, we rooted the trees with

representatives of several cyprinids subfamilies. For this

purpose, species such as Gobio gobio (Gobioninae) Rho-deus amarus, R. ocellatus and R. atremius (Acheilognati-nae), Tinca tinca (Tincinae), and Pseudorasbora parva(Gobioninae) were used as outgroups in phylogeneticanalyses.Total cellular DNA was extracted from both ethanol

preserved or frozen tissue by a standard proteinase Kand phenol/chloroform extraction method [136] andethanol purification [137]. Both mitochondrial (cyto-chrome b and COxI) and nuclear (RAG1 and S7) geneswere amplified via polymerase chain reaction (PCR)from each individual DNA sample. Primers, amplifica-tion protocols and PCR products lengths for these lociare represented in Additional file 2. In all cases, PCRmixtures were prepared under similar conditions in afinal volume of 25 μl containing 1-2 μl DNA, 0.5 μMeach primer, 0.2 mM each dNTP, 1.5 mM MgCl2, and 1unit of Taq DNA polymerase (Invitrogen). After check-ing PCR products on 1.5% agarose gels, the four geneticfragments were purified with the kit ExoSAP-IT™(USB)and directly sequenced. For RAG1 some internal pri-mers were designed to sequencing (See Additional file2). All samples were sequenced on an Applied Biosys-tems 3700 DNA sequencer following the manufacturer’sinstructions. All new are available online [Genbank:

Figure 10 Map of localities samples of the leuciscine representatives included in the phylogenetic framework. Numbers correspond tothose indicated in the Additional file 1.


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HM560056-HM560237 for cyt b; HM560238-HM560383and HM989722-HM989724 for COxI; HM560384-HM560471, HM560573-HM560586 and HM998711-HM998712 for RAG1; HM560472-HM560572 andHM998713-HM998714 for S7].

Sequences alignment and Phylogenetic AnalysesHomologous regions were aligned manually against pre-viously published cytochrome b sequences of leuciscins[4,5]. Chromatograms and alignments were visuallychecked and verified and there were no gaps in theresulting cyt b, COxI and RAG1. In these three codinggenes sequences alignment was based on the inferredamino acid sequence. Alignments sequences of all per-formed analyses are proporcioned in Additional file 3.All codon posititions were included in the analysis. Thefirst intron of S7 gene were aligned with Clustal X[138], using default parameters, to optimize sequencealignment including gaps and aligned sequences werelater checked by eye. Despite of indels are often consid-ered as a class of phylogenetic characters to be incorpo-rate in the phylogenetic analysis [139,140], gaps of S7gene was discarded in phylogenetic reconstructions dueto their ambiguity in the alignment.For all data sets, the transition (ti)/transversion (tv)

rate was estimated using a maximun-likelihoodapproach (Table 1). Furthermore, for each gene, thesaturation of transition and transversion changes waschecked by plotting the absolute number of changes ofeach codon position against patristic distances (p).There was no evidence of saturation for any data set ofsequences, even in the third position of coding genes.Analyses were performed independently on each gene

(cyt b, COxI, RAG1 and S7), in nuclear data sets and onthe total number of base pairs sequenced (4339 bp)("Total evidence”, [141]). Nucleotide composition wasexamined and the c2 homogeneity test of base frequen-cies was carried out in Paup *4.0b10 [142] for all genes.The Akaike Information Criterion implemented in Mod-elTest v. 3.7 [67] was used to determine the evolutionarymodel that best fits the data set for each data set. Themodel selected was used for subsequent analyses. Baye-sian inference (BI) was performed with MrBayes 3.1.2[143]. In the combined data set each gene partition wasallowed to follow its own model of evolution. In allcases, BI was obtained by simulating two simultaneousMarkov chain analyses (MCMC) for 3.000.000 genera-tions each, to estimate the posterior probabilities distri-bution. Topologies were sampled every 100 generationsand a majority-rule consensus tree was estimated aftereliminated the first 105 generations in each analysis.Maximum Likelihood (ML) analysis was performed withPhyML package [144]. To estimate the robustness of thelikelihood analyses a nonparametric bootstrap test was

conducted with 500 replicates. Maximum Parsimony(MP) [145] analysis was performed with the packagePAUP* 4.0b10 [142]. MP analysis was conducted withTBR branch swapping and 10 random stepwise addi-tions using the heuristic search algorithm. Analyseswere performed on each independent data set and onthe total data matrix after check homogeneity amongpartitions [146]. Confidences for this analysis were esti-mated by bootstrapping (500 repetitions) [147].

Molecular clock and divergence times estimatesAs a general clock-like behaviour was rejected, diver-gence times and their credibility intervals (highest pos-terior density: HPD) were estimated using a relaxedclock model in BEAST v1.4.7 [148], with branch ratesdrawn from an uncorrelated lognormal distribution[149] and a Yule speciation prior. Fossil evidence wasused to place the constraints of the age of differentnodes within the topology. The use of multiple calibra-tion points would provide a more realistic divergencetime estimates. The calibration points used to estimatedivergence times are represented in Table 5. Tracer v1.4[148] was used to plot the log-likelihood scores againstgeneration time to evaluate run convergence and theburn-in needed before to reconstruct the 50% majority-rule consensus tree of the post burn-in trees. To obtaina maximum clade credibility tree, trees were summar-ized with the software TreeAnnotator 1.4.6 [148].

Additional material

Additional file 1: Individuals included in the phylogeny andlocalities samples.

Additional file 2: Laboratory performance (PCR conditions andprimers).

Additional file 3: Sequences alignments used in phylogeneticperformance and molecular clock analyses.

AcknowledgementsWe thank F. Alda, P. Ornelas, C. Pedraza, A. Perdices, O. Dominguez, J. A.Carmona and I. Bogut for their help in fieldwork. L. Alcaraz assisted in the

Table 5 Fossil calibration points

Clade Mya

Squalius cephalus clade 6.5

Squalius lineage 13

Achondrostoma/Pseudochondrostoma clades 5.3

Scardinius clade 10

Scardinius erythrophthalmus clade 5.3

Leuciscus leuciscus/leuciscus idus clade 5.3

Miminun Recent Common Ancestor of all Iberian taxa 25

Abramidini clade 17

Split among Phoxinus and leuciscine groups 33


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laboratory work. N. Bogutskaya for providing samples of Leuciscus. cf. latus. A.Burton reviewed the English text. RS was supported by the projectMK00002327201 of the Czech Ministry of Culture. RS received also supportfrom the SYNTHESYS Project (ES-TAF-1187) financed by EuropeanCommunity Research Infrastructure Action under the FP6 “Structuring theEuropean Research Area” Programme”. This study was financially sponsoredby Spanish Ministry of Education and Culture project CGL2007/61231 andSpanish Agency for International Development Cooperation (AECID) project07-CAP-1523.

Author details1Museo Nacional de Ciencias Naturales-CSIC. Department of Biodiversity andEvolutionary Biology. José Gutiérrez Abascal 2, 28006 Madrid. Spain.2Senckenberg Centre for Human Evolution and Paleoecology (HEP) andInstitute for Geoscience, University Tübingen, Sigwartstr. 10, D-72076Tübingen, Germany. 3AZV Agency. Dolsko 14, S1-1262. Slovenia. 4Leibniz-Institute of Freshwater Ecology and Inland Fisheries. Müggelseedamm 310,12587 Berlin. Germany. 5National Museum. Václavské náměstí 68, 115 79Prague 1. Czech Republic. 6Istanbul University. Faculty of Science.Department of Biology, 34134 Vezneciler, İstanbul. Turkey. 7Department ofBiodiversity and Ecosystem Management, Environmental Sciences ResearchInstitute. Shahid Beheshti University G. C., Tehran, Iran.

Authors’ contributionsSP conceived the study, collected samples, obtained data and analyses anddrafted the manuscript. MB contributed to review and support thebiogeographical chapter with paleontological and fossil data. PZ, JF and RScollected samples, reviewed the manuscript and make useful suggestions forit. MO and AA collected valuables samples for the study. ID participated inits design and coordination, helped to draft the manuscript and collectedsamples. All authors read and approved the final manuscript.

Received: 25 February 2010 Accepted: 31 August 2010Published: 31 August 2010

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doi:10.1186/1471-2148-10-265Cite this article as: Perea et al.: Phylogenetic relationships andbiogeographical patterns in Circum-Mediterranean subfamilyLeuciscinae (Teleostei, Cyprinidae) inferred from both mitochondrialand nuclear data. BMC Evolutionary Biology 2010 10:265.

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